Field of the Invention
The present invention relates to the field of biological engineering and biotechnology. In particular, it relates to methods for producing L-citrulline using engineered microorganisms.
Description of the Related Art
L-arginine deiminase (Argininedeiminase, E.0 .3.5.3.6, ADI), a key enzyme of microorganism arginine metabolism pathway, catalyzes L-arginine and transforms it to L-citrulline and ammonia. ADI is widely present in Bacillus cereus, Streptococcus faecalis, Pseudomonas and mycoplasma microorganisms (e.g. malodors). It has been found that ADI could inhibit animal vascular endothelial cell proliferation and various types of malignancies proliferation. ADI purified from Mycoplasma arginine inhibits cell migration of several types of cancer cells in human body. In addition, ADI could directly control the growth of tumor cells by suppressing the synthesis of biogenic amine. Therefore, arginine deiminase as a novel anti-tumor substance has attracted attention in the field of medicine. The ADI also could be employed to the industrial production of citrulline because it can hydrolyze arginine to produce citrulline.
L-citrulline is an important non-protein amino acid, which is an important intermediate metabolite of urea cycle in a human body. It not only functions as an antioxidant by absorbing and removing harmful free radicals, but also effectively protects the DNA and PMN from oxidative reactions through increasing the arginine required for NO synthesis. In addition, it can be used as anti-aging products and health care products for improving immunity. It can also be used as anti-wrinkle and anti-aging skin care products. In the field of medicine, L-citrulline can be used to treat prostate diseases, especially for prostatitis and prostate cancer because L-citrulline enables human body to produce nitrogen oxides, which is a very important substance for male potency. Recently, studies have shown that the conversion of citrulline to L-arginine in human bodies plays an important role in maintaining the normal function of cardiovascular nitric oxide metabolism.
Methods for L-citrulline industrial production include chemical synthesis, natural extraction, microbial fermentation and L-arginine hydrolysis.
High titer strains for L-citrulline production with microbial fermentation was primarily obtained through mutagenesis or genetic engineering methods, and those strains can produce L-citrulline from cheap starting materials such as glucose and starch. These types of research started in Japan from 1930s and reached to a relatively high level in 1960s. Kyowa Hakko Kogyo Company found an Arthrobacter paraffineus that produced citrulline from hydrocarbon. It utilized 1% NH4NO3 and 0.5% yeast extract as nitrogen source combined with 1 mg/L VB1 and other ingredients, and produced 7.1g /L citrulline after 96 h fermentation. This method has disadvantages of low efficiency and long fermentation period, not suitable for L-citrulline industrial production.
Methods of L-arginine hydrolysis include alkaline hydrolysis and enzymatic hydrolysis. The method of enzymatic hydrolysis has the advantages of having higher yields, few purification steps, and few D-type optical rotation enantiomers in the final product, simple production process and low cost of the production. Ichiron Chibata reported that strains including Psudomonas putida ATCC 4359, Pseudomonas fluorescen IFO 30081, Pseudomonas ovalis IAM 1002, and Leuconostoc citrovorum ATCC 8081 produces ADI and can converse L-arginine or DL-arginine to 80 g/L citrulline. Other research has been found that 92.72g/L citrulline can be produced from arginine by Streptococcus faecalis within 25 hours. However, those methods are still far away from meeting the needs of industrial production.